Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids (or base paired regions of single-stranded nucleic acids), forming covalent adducts to pyrimidine bases upon absorption of long wave ultraviolet light (UVA). G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985); Hearst et al., Quart. Rev. Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to a psoralen-pyrimidine monoadduct and on the opposite strand, absorption of a second photon can lead to formation of a diadduct which functions as an interstrand crosslink [S. T. Isaacs et al., Biochemistry 16:1058 (1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294 (1982); J. Tessman et al., Biochem. 24:1669 (1985); Hearst et al., U.S. Pat. Nos. 4,124,598, 4,169,204, and 4,196,281, hereby incorporated by reference].
The photoreaction of psoralens with nucleic acid has been useful in the study of nucleic acid folding, the attachment of diagnostic probes to nucleic acids, the attachment of nucleic acids to surfaces and materials, the blocking of polymerase reactions and the inactivation of organisms and cells that require nucleic acid replication to proliferate, e.g., bacteria, viruses, leukocytes and overproliferating cells, such as those resulting in psoriasis, restenosis, or cancer. The inactivation of a virus can also be applied to preparation of vaccines. The level of reaction with cellular nucleic acid can be modulated to stop proliferation of the cell yet maintain cell functions such as protein synthesis. This can be applied to the treatment of T-cell lymphocytes as a means of preventing graft vs. host disease in, for example, bone marrow transplants.
The use of psoralens for pathogen inactivation in blood products is of particular interest as the safety of the blood supply is an issue of universal concern. While transfusion associated viral infections have been considerably reduced by testing, transmission of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) continue to occur in 1/450,000 to 660,000 units, 1/200,000 units and 1/3000 units respectively [R. Dodd, Blood Supply: Risks, Perceptions, and Prospects for the Future, S. J. Nance, ed., p. 1 (1994); E. Lackritz et al., New Eng. J. Med. 333: 1721 (1995)]. Testing is not an option for some viruses. Cytomegalovirus is commonly found within the blood supply yet is of clinical importance only to immune compromised patients for which infection can be fatal [R. Bowden, Blood Safety: Current Challenges, S. J. Nance, ed., p.201 (1992)]. Universal screening for CMV would lead to a serious reduction in eligible donors and thus a reduction in the national blood supply. Special donor pools must be used for these patients at present. It is also recognized that other unknown viruses or new strains of known viruses may find their way into the blood supply and will not be identified until morbidity or mortality is noted, nor will they be able to be screened out until tests become available. The identification of hepatitis G in blood units is the most recent example of such an occurrence [H. Alter, Transfusion 37: 569 (1997)].
Bacterial contamination, especially of platelet concentrates (PC) has been increasingly recognized as a problem as well. It is estimated that 1/1,000 to 2,000 PC units show levels of contamination that results in a septic response [J. Morrow et al., JAMA 266: 555 (1991); M. Blajchman, Blood Safety: Current Challenges, S. J. Nance, ed., p.213 (1992); E. Chiu et al., Transfusion 34: 950 (1994)]. There are at present no screening tests available for blood units for any of the ten or so bacteria that have been associated with fatal transfusion associated sepsis in the United States. While there are potential methods for storage of platelets at lower temperatures to alleviate this problem [U.S. Pat. Nos. 5,827,640 and 5,827,741], inactivation of the bacteria by psoralen would have far less impact on the routine storage of platelets.
Psoralens are ideal candidates for photosensitized, decontamination of platelet concentrates [H. Alter et al., Lancet ii:1446 (1988); L. Lin et al., Blood 74: 517 (1989); C. Hanson, Blood Cells 18: 7 (1992)]. For example, 8-methoxypsoralen (8-MOP) is quite effective at deactivation of a number of bacteria found in platelet concentrates. However, it is not sufficiently active to inactive pathogens with small genomes (i.e., viruses) without using concentrations and irradiation times which damage platelets. The highly active psoralen, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), exhibits excellent photochemical inactivation properties but is highly mutagenic in the absence of light in some bacterial assays [S. Wagner et al., Photochem. Photobio. Meeting Abstract, 55: 113S (1992)]. Other 4'- and 5'-aminomethyl substituted psoralens have been developed which show excellent photochemical inactivation properties with considerable reduction in mutagenicity.
Several patents are directed toward psoralen inactivation of pathogens in blood products [G. Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and 4,748,120, L. Lin et al., U.S. Pat. Nos. 5,288,605, 5,482,828, and 5,709,991, and S. Wollowitz et al., U.S. Pat. No. 5,593,823, hereby incorporated by reference]. P. Morel et al., Blood Cells 18:27 (1992) show that 300 .mu.g/mL of 8-MOP together with ten hours of irradiation with ultraviolet light can effectively inactivate viruses in human serum. Similar studies using 8-MOP and AMT have been reported by other investigators [Dodd R Y, et al., Transfusion 31:483-490 (1991); Margolis-Nunno, H., et al., Thromb Haemostas 65: 1162 (Abstract)(1991)]. Indeed, the photoinactivation of a broad spectrum of microorganisms has been established, including HBV, HCV, and HIV. [Hanson C. V., Blood Cells: 18: 7-24 (1992); Alter, H. J., et al., The Lancet ii:1446 (1988); Margolis-Nunno H. et al., Thromb Haemostas 65: 1162 (Abstract) (1991); Lin et al. Transfusion 37: 423 (1997)]. There are clearly a broad class of psoralen compounds effective in the inactivation of pathogens in general and particularly in blood products.
The most highly active psoralen compounds useful for inactivation have amino derivatives on the 4' and 5' positions [Wollowitz et al., U.S. Pat. Nos. 5,578,736 and 5,654,443, Kaufman U.S. Pat. No. 4,294,822]. 5-alkoxy and 8-alkoxypsoralens with amino substituents at the 8 or 5 position, respectively, as well as 8-aminomethyl psoralen and 8-aminomethyl-4-methylpsoralen are known [J. Hansen et al., J. Med. Chem. 28: 1001-1010 (1985); Kaufman U.S. Pat. Nos. 4,269,851 and 4,328,239]. The limited data provided for these latter compounds suggested that even the amino substituted alkoxypsoralens have relatively poor photoactivity and that amino substitution at the furan ring is important for high photoactivity. Also, these compounds are formed by methods which offer little flexibility in modifying the ring functionality.